Among post-translational modifications, protein phosphorylation is particularly relevant to cancer biology and therapy. However, despite advances in proteomics, it is still difficult to pinpoint phosphorylation sites in proteins. The long-term goal of this project is to develop and commercialize a multiplexed method for isolating, identifying, and quantifying phosphorylation sites using phosphorylation-specific antibodies. This method would contribute to the development of a new generation of drugs tailored to inhibit specific protein kinases with roles in cancer by identifying new phosphorylation sites that could become targets for cancer diagnosis and treatment. During our IMAT-funded Phase I, we established the feasibility of using an immunoaffinity method to isolate phosphopeptides from complex mixtures, which were then identified by tandem mass spectrometry. In this Phase II application, we will optimize the immunoaffinity method so it can be used to analyze low-level samples, using cell numbers that are comparable to what would be available from patient samples. Once we have optimized the method and established the repertory of antibodies that can be used productively in the method, we will demonstrate the method's utility by applying it to a variety of cancer cell lines, to show the method can probe the major signal transduction networks involved in cancer and can identify oncogenic lesions. We will bring a quantitative dimension to the method by merging it with mass spectrometry methods such as SILAC and Aqua, which are needed for later biomarker discovery and biomarker assay efforts.